Heart valves play a critical role in cardiac function by promoting unidirectional blood flow and are a frequent target of congenital heart disease. Atrioventricular valve formation is triggered by signals that are precisely localized to the junctin between chambers (also known as the atrioventricular canal or AVC). These signals instruct the local endocardium to bulge into the lumen of the heart tube, forming structures known as cardiac cushions that later remodel into valve leaflets. Studies in zebrafish have shown that canonical Wnt pathway activity at the AVC promotes the expression of genes encoding Bmp signals that induce cushion formation. Proper heart development necessitates the restriction of these inductive signals to the AVC, yet we do not understand the mechanisms responsible for this confinement. We also do not know important general properties of this system, such as whether Wnt signaling patterns itself or if it relays an upstream pattern. I will pursue two specifc aims to expand our knowledge of the components and systems-level properties of the AVC patterning network. My first aim investigates serotonin signaling, which our preliminary data suggest has a critical role in AVC patterning. Treatment of embryos with the serotonin receptor antagonist ketanserin results in ectopic bmp4 expression beyond the boundaries of the AVC. This phenotype is recapitulated by knockdown of the serotonin receptor htr2a, suggesting that serotonin signaling through this receptor is essential. I hypothesize that serotonin signaling participates in the confinement of cushion-inducing signals to the AVC. To test this hypothesis, I will first examine a panel of markers to thoroughly characterize the cushion patterning defect in htr2a-deficient embryos. I will next use pharmacological perturbations to determine the time window when serotonin signaling is critical for AVC patterning. I will also examine Wnt pathway readouts and perform epistasis experiments to determine whether serotonin acts upstream or downstream of the Wnt pathway to restrict cushion induction. Finally, I will use pharmacological perturbations of global serotonin levels in combination with htr2a misexpression to determine whether serotonin signaling is instructive or permissive for cushion restriction. In my second aim, I will test the hypothesis that Wnt signaling patterns itself, i.e. self- organizes. Self-organizing systems have several useful properties such as stability and robustness to fluctuations. These systems often contain a self-amplifying component (positive feedback) that is balanced by inhibition generated either by itself (autoinhibition) or by a rival self-amplifying component that dominates an adjacent domain (inhibitory crosstalk). I will use a combination of novel optogenetic techniques and live signaling reporters to determine whether Wnt signaling in the AVC displays hallmark characteristics of self-organizing systems, such as positive feedback, autoinhibition, and inhibitory crosstalk. Together, these studies will reveal important new mechanistic information and systems-level properties of the AVC patterning network, which will help us to understand the etiology of congenital heart defects.